This invention relates to a low impedance cell potential measuring electrode assembly typically having a number of microelectrodes on an insulating substrate and having a wall enclosing the region including the microelectrodes. The device is capable of measuring electrophysiological activities of a monitored sample using the microelectrodes while cultivating those cells or tissues in the in the region of the microelectrodes. The invention utilizes independent reference electrodes to lower the impedance of the overall system and to therefore lower the noise often inherent in the measured data. Optimally the microelectrodes are enclosed by a physical wall used for controlling the atmosphere around the monitored sample.
Cell potential measuring apparatus have been developed to measure the activity or electrical potential generated by activity of nerve cells, other cells, or tissues (for example, Japanese Kokai 8-62209) without inserting glass electrodes or the like into the cells.
Measurement of cell potential by inserting a glass electrode or the like into the cell may damage that cell. Long term measurement of cell potential is quite difficult. It is further difficult to measure plural positions simultaneously; there is a limit to the number of electrodes one can place in a measurement electrode array and it is similarly difficult to adequately determine the position of the sample over measurement electrodes. In contrast, use of a cell potential measuring electrode having plural microelectrodes on a substrate (having a wall for enclosing a region including the microelectrodes), allows cultivation of the cells within the region enclosed by the wall and the simultaneous measurement of the potential of plural positions without damaging those cells.
These cell potential measuring devices measures cell potential against a reference. One such way is discussed with regard to Kokai 8-62209. When 64 microelectrodes are arranged in eight columns and eight rows, theoretically, by using one microelectrode as the reference potential (that is, as a common reference electrode connected to the potential of the culture medium) the cell potential of the other 63 positions can be measured simultaneously by using the remaining 63 microelectrodes.
However, when measuring very low level or micro-potentials such as cell potentials, noise is a problem. Noise level varies significantly depending on the selection of the type and location of the reference electrode. As mentioned above, when using one microelectrode as a reference electrode, simultaneous measurement of potential at 63 positions by using the remaining 63 microelectrodes is impossible because of the high noise level. When the reference electrodes and measuring electrodes correspond one-by-one to each other, the potential may be measured at a very low noise level state; But if 64 microelectrodes are used, for example, corresponding to 32 reference electrodes and 32 measuring electrodes, only 32 positions can be measured simultaneously.
In theory, though, one must limit the number of reference electrodes in order to simultaneously measure the potential at as many positions as possible.
As shown in FIG. 12, eight microelectrodes in one row are used as reference electrodes and seven measuring electrodes each are correlated to each of the reference electrodes, so that the potential can be measured simultaneously at 7xc3x978=56 positions. If 56 microelectrodes are used as measuring electrodes, i.e., by using eight microelectrodes in one row as reference electrodes, the loss of measuring sites is about 12% as compared with the case of using all 64 or 63 pieces as measuring electrodes. However, even when seven measuring electrodes are used with one reference electrode, the noise is still quite large. It is quite difficult to detect a small change in cell potential from the noise.
Moreover, as shown in FIG. 12, when placing a segment S of cell or tissue on the plural microelectrodes, the segment S should not be placed on the row of microelectrodes used as reference electrodes. Such a placement requires skill and is difficult because the segment S must be held by tweezers and moved while observing the segment through a microscope. It is extremely difficult to place the segment S so that the eight microelectrodes in one row are completely exposed, while the remaining 56 microelectrodes be completely covered with the segment. If the segment S is placed to completely expose the eight microelectrodes in one row, usually some of the remaining 56 are exposed, and hence the number of positions for simultaneous measurement is decreased.
The invention is intended to solve such problems. This invention provides a cell potential measuring electrode less susceptible to noise and is yet capable of simultaneously measuring the potential at many positions by effectively utilizing all of the available microelectrodes if the positioning is not very precise when placing the segment of cell or tissue to be measured.
The cell potential measuring electrode of the invention preferably includes plural microelectrodes on an insulating substrate, a conductive pattern for connecting the microelectrodes to some region out of the microelectrode are, electric contacts connected to the end of the conductive pattern, an insulating film covering the surface of the conductive pattern, and a wall enclosing the region including the microelectrodes on the surface of the insulating film. The inventive reference electrodes have a comparatively lower impedance than the impedance of the measuring microelectrodes. They are respectively placed at plural positions in the region enclosed by the wall and often at a specific distance from the microelectrodes. The electrical contacts are further usually connected between the conductive pattern for wiring of each reference electrode and the end of the conductive pattern. The surface of the conductive pattern for wiring of the reference electrodes is typically covered with an insulating film.
According to this invention, since exclusive reference electrodes are provided at plural positions distant from the region of plural measurement microelectrodes, it is easy to place the segment of cell sample to cover all microelectrodes while not contacting with the reference electrodes. The reference electrode would typically have, for example, a larger area than a measurement microelectrodes and hence is smaller in impedance. Therefore the noise level is small even if connected to plural reference potentials for measuring positions. Therefore, common reference electrodes can be used with multiple measurement microelectrodes. Moreover, since each one of the plural reference electrodes is responsible for multiple measurement microelectrodes, the cell potentials may be easily measured simultaneously using all of microelectrodes.
Preferably, the plural reference electrodes are placed at nearly equal distances from the plural microelectrode region and at intervals of nearly equal angle. By xe2x80x9cintervals of nearly equal anglexe2x80x9d, we mean that when the plural microelectrode region is viewed from above, the plural reference electrodes extend away from that region in equi-angular rays. More preferably, the plural microelectrodes are placed in a rectangular matrix, and four of the reference electrodes are provided on an extension of diagonals of the region holding that rectangular matrix. In such a symmetrical placement, the noise level to each microelectrode is averaged.
As a specific example, the microelectrodes are situated in a matrix arrangement in a rectangle having sides of, e.g., 0.8 to 2.2 mm (in the case of 450 xcexcm microelectrode pitch) or 0.8 to 3.3 mm (in the case of 300 xcexcm microelectrode pitch). Four reference electrodes are situated at four corners of a rectangle of 5 to 15 mm on one side. More preferably, 64 microelectrodes are disposed in eight rows and eight columns at central pitches of about 100 to 450 xcexcm, preferably 100 to 300 xcexcm.
In order to set the impedance of the reference electrodes to be sufficiently smaller than the impedance of the microelectrodes, the area of the reference electrodes is preferably 4 to 25 times (particularly preferably 16 times) the area of the microelectrodes. As a specific example, the area of each of the microelectrodes is preferably between about 4xc3x97102 and 4xc3x97104 xcexcm2 and the area of each of the reference electrodes is preferably between about 64xc3x97102 and 64xc3x97104 xcexcm2. 
Preferably the microelectrodes and the reference electrodes are formed from the same material to both simplify the manufacturing process and obtain a cost benefit. Preferably, the microelectrodes and the reference electrodes are formed of layers of nickel plating, gold plating, and platinum black on an indium-tin oxide (ITO) film. After platinization, the impedance of the reference electrodes is preferably between 2 and 3 kilohms.
The insulating substrate (for example, a glass substrate) may be nearly square. Plural electric contacts may be connected to the end of the conductive pattern and preferably are placed on the four sides of the insulating substrate. As a result, layout of wiring patterns of multiple microelectrodes and reference electrodes is easy. Because the pitches of electric contacts may be made to be relatively large, electric connection through the electric contacts with external units is also easy.
The microelectrode region is usually very small. When observing the sample through a microscope, it is hard to distinguish position and both vertical and lateral directions. It is desirable to place indexing micro-marks near the microelectrode region to allow visual recognition through the microscope variously of direction, axes, and position.
The most preferred cell potential measuring apparatus of this invention is made up of a cell placement device having cell potential measuring electrodes, contact sites for contacting with an electric contact, and an electrode holder for fixing the insulating substrate by sandwiching from above and beneath. In a variation of the invention, a signal processor may be placed near the microelectrode matrix or region. The cell potential measuring electrodes may be connected electrically to the cell placement assembly device to allow processing of the voltage signals generated by the sample and measured between each such microelectrode and the reference electrodes. The cell potential measuring assembly usually includes a region enclosed by a wall for cultivating sample cells or tissues. It also preferably includes an optical device for magnifying and observing optically the cells or tissues cultivated in the region enclosed by the wall. This cell potential measuring apparatus preferably further comprises an image memory device for storing the magnified image obtained by the optical device.